Breast cancer is the most common cancer in women. Most breast cancers are detected early and treated with multimodality therapy including surgery, chemotherapy and radiation therapy. Despite patients being rendered disease free with this intensive therapy, many women with high risk features will have recurrent disease. Over expression of the Her-2/neu protein is one such high risk feature.
The epithelial cell adhesion molecule, Her-2/neu is a member of the epidermal growth factor receptor family, normally expressed during fetal development. Amplification of the gene and over-expression of the protein product have been described in a number of epithelial tumors and are markers of high recurrence risk in breast cancer. Immunotherapy directed against Her-2/neu can control the growth of these tumors. The Her-2/neu protein is over-expressed in 30-40% of early stage breast cancer and that over-expression is associated with poor clinical outcome. Other cancers in which Her-2/neu is over-expressed include ovary, recto-colon, lung, prostate, stomach, pancreatic, and biliary cancer (Sotiriadou, N. N., et al. Cancer Immunology Immunotherapy, in press (online Sep. 8, 2006)).
The Her-2/neu protein is also a source of immunogenic peptides. Immunogenic peptides of Her-2/neu can stimulate cytotoxic T lymphocytes (CTL) to recognize and kill Her-2/neu expressing cancer cells in vitro. Some of the peptides (E75 and GP2) are being used in clinical trials as vaccines in patients with Her-2/neu+ breast cancers. Thus far, they have been shown to be safe in patients and effective in stimulating antigen specific immunity; more importantly, we have shown that the immunity conferred by E75 appears to have clinical benefit in preventing recurrence of breast cancer. Unfortunately, the immunity conferred by these peptide vaccines is not sustained. Helper peptides may be required to increase the efficiency of induction and establishment of long-term immunity.
Helper peptides for Her-2/neu have been described. The MHC Class II associated Ii protein normally blocks the processing of endogenous peptides, preventing attachment to MHC and antigen presentation. Suppression of the Ii protein in tumor cell lines and rat tumor models has been shown to induce tumor antigen presentation and enhance antigen specific tumor cell killing.
The Ii protein normally binds to MHC class II molecules in the endoplasmic reticulum at synthesis and protects the epitope-binding site on MHC class II molecules from binding to endogenously-derived epitopes in the endoplasmic reticulum, as normally occurs with MHC class I molecules [Bertolino, P. and Rabourdin-Combe, C., Crit. Rev Immunol 16:359-379 (1996); Bodmer, H., et al., Science 263:1284-1286 (1994)]. Another major function of the Ii protein is to enhance exogenous peptide charging to MHC class II molecules [Xu, M., et al., Mol Immunol 31:723-731 (1994); Daibata, M., et al., Mol Immunol 31:255-260 (1994); Reyes, V. E., et al., Ann N Y Acad Sci 730:338-341 (1994)]. The MHC class II/Ii complex is transported to a post-Golgi, antigenic peptide-binding compartment after synthesis [Bakke, O. and Dobberstein B., Cell 63:707-716 (1990); Lamb, C. A. and Cresswell, P., J Immunol 148:3478-3482 (1992); Blum, J. S, and Cresswell, P., Proc Natl Acad Sci USA 85:3975-3979 (1988)]. In such compartments, the Ii is cleaved by proteases to allow charging by exogenously derived epitopes. After being charged with epitopes, the MHC class II/epitope complex travels to the cell surface for presentation to CD4+ Th cells [Nguyen, Q. V et al., Hum Immunol 24:153-163 (1989); Shi, G. P., et al., J Exp Med 191:1177-1186 (2000); Riese, R. J., et al., Immunity 4:357-366 (1996); Hiltbold, E. M. and Roche, P. A., Curr Opin Immunol 14:30-35 (2002)]. Two mechanisms have been proposed to explain the function of Ii in enhancing the charging of epitopes to MHC class II molecules. First, Ii is partially digested to leave only a small segment behind, termed CLIP, which is bound to the epitope-binding groove of the MHC class II molecule in a manner to keep the groove open [Riberdy, J. M., et al., Nature 360:474-477 (1992); Gautam, A. M., et al., Proc Natl Acad Sci USA 92:335-339 (1995); Romagnoli, P. and Germain, R. N., J Exp Med 180:1107-1113 (1994)]. HLA-DM then exchanges CLIP for an epitope [Morris, P., et al., Nature 368:551-554 (1994); Denzin, L. K. and Cresswell, P., Cell 82:155-165 (1995)]. Secondly, in a concerted manner, Ii is digested and released from MHC class II molecules as epitopes are being charged [Xu, M., et al., Mol Immunol 31:723-731 (1994); Daibata, M., et al., supra; Reyes, V. E., et al., Ann N Y Acad Sci 730:338-341 (1994)].
An important function of the Ii protein is evident in a short sequence that binds to an allosteric site outside of the epitope-binding groove [Xu, M., et al., Arzneimittelforschung 49:791-799 (1999)]. The result of this interaction is the maintenance of the epitope-binding groove in a conformation that is the most suitable for MHC class II molecules to exchange an epitope. That Ii sequence has been termed the Ii-Key peptide. The segment of the Ii containing amino acids hIi(77-92) regulates tightness of closure of the antigenic epitope-binding groove of MHC class II molecules. This segment first raised interest due to it's having 6 positive side chains, no negative side chains, and 4 prolines, which together appeared to constitute a signal for a protease or “exchange-ase”; ostensibly to regulate cleavage and release of the Ii [Adams, S., et al., Arzneimittelforschung 47:1069-1077 (1997); Lu, S., et al., J Immunol 145:899-904 (1990)]. Further studies showed that mutations in this segment do, in fact, block the staged cleavage and release of Ii [Xu, M., et al., Mol Immunol 31:723-731 (1994); Daibata, M., et al., supra]. In light of these findings, we synthesized the fragment of the Ii containing amino acids hIi(77-92), referred to as “Ii-Key”. An initial study illustrated that the activation of hen egg white lysozyme (HEL)-specific T cell hybridoma by an HEL epitope peptide was enhanced by Ii-Key peptides up to 50-fold [Adams, S., and Humphreys, R. E., Eur J Immunol 25:1693-1702 (1995)]. Enhanced activation was observed even when using paraformaldehyde-fixed Antigen Presenting Cells (APCs), in which normal intracellular processing was not possible. Studies to further identify the minimal active sequence of Ii-Key revealed a ‘core’ LRMKLPK (SEQ ID NO: 1) structure that had greater potency than the original 16-amino acid peptide [Adams, S., et al., supra]. The Ii 77-80 (LRMK (SEQ ID NO: 2)) segment retained at least 50% of the activity of LRMKLPK (SEQ ID NO: 1). For simplicity, we therefore designed later Ii-Key/MHC class II epitope hybrids with this shorter, four-amino-acid, Ii-Key moiety.
In contrast to cell culture studies, in vivo inoculation of mice with Ii-Key plus an antigenic peptide failed to enhance the activity of that antigenic peptide [unpublished observations]. This suggested that the Ii-Key needed to be co-localized with the antigenic epitope to enhance presentation. The Ii-Key moiety was linked covalently to the MHC class II epitope to ensure that individual MHC class II molecules on the APC are exposed simultaneously to both Ii-Key and epitope. A systematic series of Ii-Key MHC class II epitope hybrids were synthesized and tested in an in vitro T cell hybridoma stimulation assay [Humphreys, R. E., et al., Vaccine 18:2693-2697 (2000)]. For this series of hybrids, the Ii-Key core (LRMK (SEQ ID NO: 2)) was joined to an MHC class II-restricted epitope of pigeon cytochrome C (PGCC81-104). The spacers joining Ii-Key and PGCC81-104 were either a simple polymethylene (δ-aminovaleric acid; ava) linker or the natural sequence of the Ii extending from the C-terminus of LRMK (SEQ ID NO: 2). The design of Ii-Key hybrids was based on biochemical and X-ray crystallographic data indicating that the Ii-Key binding site lies outside of the antigenic peptide-binding groove of MHC class II molecules [Ghosh P, et al., Nature 378:457-462 (1995)]. Both the length of the Ii-Key derivative and linker composition were varied within the series. Hybrids having either type of bridge were effective. Some hybrids enhanced presentation of an antigenic epitope up to 250 times above the baseline stimulation observed using the free antigenic peptide [Humphreys, R. E. et al., supra].
The discovery of numerous clinically relevant peptide epitopes, both MHC class I- and II-restricted, has increased the motivation to develop effective peptide vaccines. From these data, consensus motifs for both MHC class I- and II-restricted epitopes have been proposed [Stevanovic, S, and Rammensee, H. G., Behring Inst Mitt: 7-13 (1994); Rammensee, H. G., et al., Immunogenetics 41:178-228 (1995); Hakenberg, J., et al., Appl Bioinformatics 2:155-158 (2003)]. While some peptides of potential clinical importance have been identified and specific immune responses have been observed in patients treated with those peptides, good therapeutic efficacy has not been observed [Knutson, K. L., et al., Clin Cancer Res 8:1014-1018 (2002); Phan, G. Q., et al., J Immunother 26:349-356 (2003); Brinkman, J. A., et al., Expert Opin Biol Ther 4:181-198 (2004); Hersey, P., et al., Cancer Immunol Immunother 54:208-218 (2005)]. The main obstacle appears to be the relatively low affinity of some MHC class II-restricted epitopes. There is a need for peptide vaccine potency to breakdown tolerance to tumor antigens.
A novel technology has been developed based on using a portion of the Ii protein to enhance MHC class II epitope charging and thus the efficiency of Th cell activation [Adams, S., and Humphreys, R. E., supra; Adams, S., et al., supra; Xu, M., et al., Arzneimittelforschung 49:791-799 (1999); Xu, M., et al., Scand J Immunol 54:39-44 (2001)]. This “Ii-Key” segment of the Ii protein significantly enhances MHC class II epitope presentation in a variety of settings and creates a practical method to enhance the efficacy of MHC class II peptide vaccines. The Ii-Key segment binds an allosteric site on MHC class II molecules to loosen their epitope-binding groove, allowing the epitope segment to directly charge MHC class II molecules present on the cell surface [Adams, S., et al., supra; Xu, M., et al., Arzneimittelforschung 49:791-799 (1999)]. In-Key hybrids are composed of the Ii-Key moiety linked to the N-terminus of an MHC class II epitope. This linkage can be of several forms, including a simple polymethylene bridge or the natural sequence of Ii extending from the C-terminus of LRMK (SEQ ID NO: 2) or natural amino acids (extending from the N-terminal of the MHC class II epitope) of the protein from which the MHC class II epitope peptide was obtained.
Ii-Key is a 4-amino-acid motif that has been reported to increase helper epitopes' occupancy of the MHC class II molecules and enhance CD4 T cell responses. Ii-Key hybrids are much more potent than epitope-only peptides, both in vitro and in animal studies in vivo, when used in conjunction with epitopes relevant to different diseases [Humphreys, R. E. et al., supra; Gillogly, M. E., et al., Cancer Immunol Immunother 53:490-496 (2004); Kallinteris, N. L., et al., Vaccine 21:4128-4132 (2003); Kallinteris, N. L., et al., Vaccine 23:2336-2338 (2005); Kallinteris, N. L., et al., J Immunother 28:352-358 (2005)]. In vitro, some hybrids have been shown to enhance presentation of an antigenic epitope up to 250 times above the baseline stimulation observed using the free antigenic peptide [Humphreys, R. E., et al., supra]. By enhancing the ability of peptide epitopes to charge MHC class II molecules directly on the cell surface, Ii-Key hybrid technology opens the door to a potent and clinically practical strategy for peptide immunotherapy.
Another challenge specific to cancer immunotherapy is that tumor antigens are usually tolerated as self by the immune system. Therefore, the main task of clinical immunologists is to break down tolerance to specific, tumor-associated self-antigens [Touloukian, C. E., et al., J Immunol 164:3535-3542 (2000); Knutson, K. L et al., supra; Phan, G. Q., et al., supra; Brinkman, J. A., et al., supra; Hersey, P., et al., supra]. We propose that the ability of Ii-Key hybrids to enhance activation of Th1 CD4+ cells will help greatly to break tolerance to tumor antigens. In our in vitro and in vivo animal studies using Ii-Key/gp100(46-58) and Ii-Key/HER-2/neu(777-789) hybrids [Kallinteris, N. L., et al., Vaccine 23:2336-2338 (2005); Kallinteris, N. L., et al., Frontiers in Bioscience: 46-58 (2006)], significantly stronger CD4+ T cell activity was obtained. Furthermore, the use of Ii-Key hybrids with inflammatory cytokines or adjuvants is expected to enhance the activity of hybrids and likewise help to breakdown tolerance against tumor antigens.
In order to assess the activity of Ii-Key hybrids in human cells, an Ii-Key/HER-2/neu(777-789) epitope hybrid was used to stimulate lymphocytes from both a healthy donor and a patient with HER-2/neu positive metastatic breast carcinoma. The in vitro proliferation and Interferon (IFN)-γ release was more strongly stimulated by the Ii-Key hybrid than by the epitope-only peptide [Gillogly, M. E et al., supra]. Subsequent studies, using Peripheral Blood Mononuclear Cells (PBMC) from more than 20 patients with HER-2/neu-positive cancer, have confirmed the increased T helper activity of Ii-Key/HER-2/neu hybrids relative to epitope-only peptide in stimulating CTL effectors [Sotiriadou, N. N., et al. Cancer Immunology Immunotherapy, in press (online Sep. 8, 2006)].
Life threatening diseases such as cancer demand new efforts toward effective vaccine design. Peptides represent a simple, safe, and adaptable basis for vaccine development. However, the potency of peptide vaccines is insufficient in most cases for significant therapeutic efficacy. The discovery of Ii-Key is of significant importance in designing potent peptide vaccines. The MHC class II/epitope complex is relatively stable [Fleckenstein, B., et al., Semin Immunol 11:405-416 (1999); Joshi, R. V., et al., Biochemistry 39:3751-3762 (2000)]. Ii-Key (LRMK (SEQ ID NO: 2)) facilitates the direct loading of epitopes to the MHC class II molecule groove. Without Ii-Key, peptide epitopes have difficulty displacing the pre-bound ambient peptides on MHC class II molecules at the cell surface. With the help of Ii-Key, however, the peptide-binding groove on MHC class II molecules can be opened and closed easily, offering an efficient method to enhance the binding of vaccine peptides to MHC class II molecules. Linking the Ii-Key moiety to an MHC class II epitope, to generate an Ii-Key/MHC class II epitope hybrid, greatly enhances the vaccine potency of the tethered epitope.
This type of vaccine development technology could greatly benefit tumor immunotherapy. Peptides represent the safest form of all vaccine modalities, as they are comprised of the minimal elements required for generation of an effective immune response: MHC class I and/or class II epitopes. However, while immune responses have been observed using peptide vaccines, the demonstration of clinical efficacy is rare, pointing to the need for increased potency. Although peptide vaccine research initially focused on MHC class I epitopes to induce CTL activity, MHC class II epitope vaccines for the induction of Th cell activity have drawn growing attention [Wang, R. F., Immunol Rev 188:65-80 (2002); Sette A., and Fikes, J., Curr Opin Immunol 15:461-470 (2003); Hanson, H. L. et al., J Immunol 172:4215-4224 (2004); Wong, R., et al., Clin Cancer Res 10:5004-5013 (2004)]. Recent data has clearly shown that CD4+ Th cell activation is required for the induction of a potent immune response against an immunogen. Antigen-specific Th cells are needed for full activation of antigen-specific CD8+ CTLs and to provide long-term antigen-specific memory [Hung, K., et al., J Exp Med 188:2357-2368 (1998); Surman, D. R., et al., J Immunol 164:562-565 (2000); Welsh, R. M., et al., Annu Rev Immunol 22:711-743 (2004)].
CD4+ Th cells play a critical role by inducing and maintaining both CD8+ T cell and B cell responses and in maintaining immunological memory [Hung, K., et al., J Exp Med 188:2357-2368 (1998); Surman, D. R., et al., J Immunol 164:562-565 (2000); Welsh, R. M., et al., Annu Rev Immunol 22:711-743 (2004), Knutson, K. L. and Disis, M. L., Cancer Immunol Immunother (2005); Rocha B. and Tanchot, C., Curr Opin Immunol 16:259-263 (2004); Janssen, E. M., et al., Nature 434:88-93 (2005); Dissanayake, S. K., et al., Cancer Res 64:1867-1874 (2004)]. For example, F. Ossendorp et al. established that tumor antigen-specific Th cells are required for optimal induction of CTLs against MHC class II-negative tumors [J Exp Med 187:693-702 (1998)]. The role of CD4+ Th cells in cancer immunity is further highlighted by significant clinical results obtained in melanoma patients receiving adoptive transfer of highly reactive CD8+ and CD4+ T cells [Dudley, M. E., et al., Science 298:850-854 (2002); Robbins, P. F., et al., J Immunol 169:6036-6047 (2002)]. F. G. Gao et al. showed that in order to activate memory CD8+ T cells to become fully functional tumor killer cells, antigen-specific CD4+ Th cells were required [Cancer Res 62:6438-6441 (2002)]. P. Yu et al. defined how the complementary role of CD4+ Th cells is required for efficient cross-presentation of tumor antigens to CD8+ T cells [J Exp Med 197:985-995 (2003)]. Furthermore, CD4+ T cells can help to break down tolerance to persistent self-antigens (e.g., tumor-associated antigens) to fight established tumors in an Interleukin (IL)-2 dependent mechanism [Anthony, P. A., et al., J Immunol 174:2591-2601 (2005)]. Along with the continuing discovery of novel defined epitopes, the investigation of MHC class II epitope-based vaccines in tumor immunotherapy is advancing [Wang, R. F., supra; Sette A., and Fikes, J., supra; Hanson, H. L. et al., supra; Wong, R., et al., supra; Mandic, M., et al., J Immunol 174:1751-1759 (2005); Lu, J., et al., J Immunol 172:4575-4582 (2004); Slager, E. H., et al., J Immunol 172:5095-5102 (2004)].
Conventional peptide vaccines have a number of disadvantages. First, they do not stimulate CD4+ T lymphocyte responses, which leads to a lack of B cell response and a lack of immunologic memory. Second, they are not well processed by the immune system. Therefore, clinically, these vaccines require coadministration with immune stimulants such as Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF). More importantly, the vaccines are of limited use, restricted to only those patients with HLA A-2 subtype, an HLA phenotype which occurs only in about 50% of the population but which most efficiently processes these peptides for antigen expression.
Several methods such as LEAPS and ISCOMATRIX have been developed to enhance the potency of peptide vaccines. A variety of techniques have been explored to improve the activity of peptide vaccines but none are MHC class II epitope-specific. These methods use different mechanisms for peptide vaccine enhancement. Improving the delivery of MHC class II epitopes into APCs is a common approach to enhancing MHC class II peptide vaccines. For example, exosomes are used as a delivery vehicle for better activation of both CD8+ and CD4+ T cells [Delcayre, A. and Le Pecq, J. B., Opin Mol. Ther. 8:31-8 (2006)]. Another method is ISCOMATRIX which is a cage-like structure composed of antigens, such as peptides, and adjuvants [Sanders, M. T., et al., Immunol Cell Biol. 83:119-28 (2005)]. ISCOMATRIX effectively induces both humoral and cellular immune responses against the antigens incorporated in the structure by enhancing the delivery of peptide antigens and by providing adjuvant stimulation. APCs usually have difficulty acquiring soluble antigen. Antigen/antibody complexes are more accessible to APCs through the recognition of the Fey receptor on APCs by the Fc domain on an antibody [Nagata, Y., et al., Proc Natl Acad Sci USA 99:10629-34 (2002)]. Molecular chaperones are necessary components for better binding of epitopes to MHC class II molecules. Bacterial HSP 70 enhances the immune response against MHC class II epitopes complexed to bacterial HSP70 [Tobian, A. et al., J Immunol 172:5277-86 (2004)]. However, the enhancement occurs only at low pH, indicating that chaperone-complexed epitope binding to MHC class II is also limited by CLIP [Urban, R. G., et al., Exp Med 180:751-755 (1994)]. More recently, peptide conjugates targeting specific components of immune cells have been investigated. An example of the latter employs a T cell-binding ligand coupled to a peptide antigen (Ligand Epitope Antigen Presentation System, LEAPS) [Zimmerman, D. H. and Rosenthal, K. S., Front Biosci 10:790-798 (2005)]. Because Ii-Key hybrids target the charging of MHC class II molecules, antigen presentation to T cells is more selective than LEAPS technology. Peptide vaccines are usually limited by polymorphic MHC class II allele restrictions. A non-specific T helper epitope technology, the pan-DR epitope (PADRE), has also been developed to circumvent this limitation [Franke, E. D., et al., Vaccine 17:1201-5 (1999)]. The advantage of PADRE is that it overcomes restrictions by HLA-DR alleles. DNA vaccines using the Ii gene as a delivery carrier to deliver T helper epitope to MHC class II molecules have been developed [Nagata, T et al., Vaccine 20:105-14 (2001)]. This method utilizes an Ii gene in which the CLIP portion has been replaced by a DNA fragment encoding a MHC class II epitope. This method has successfully induced epitope-specific CD4+ T cells. It should also be noted that Ii-Key hybrid technology is compatible with many of the methods discussed to further enhance the overall antigen-specific response. For example, Ii-Key hybrid peptides might be incorporated into ISCOMATRIX to further enhance the potency of the vaccine.
A hybrid peptide of a Her-2/neu antigenic epitope p776-790 and the Ii-Key protein has been developed. This hybrid protein, called AE37, brings the antigenic epitope into close proximity of the target binding site on class II MHC molecules, thus priming the molecule for antigen presentation. We have performed a phase Ib trial of the AE37 peptide vaccine in human Her-2/neu+ breast cancer patients to document toxicity and measure immunologic responses to escalating doses of the vaccine. The results of this trial are presented here.